A strain of Trichoderma reesei genetic engineering bacteria with high cellulase production induced by soluble and insoluble carbon sources and its construction method and application

A technology of genetically engineered bacteria and Trichoderma reesei, applied in the biological field, can solve the problems of low β-glucosidase yield and low cellulase yield, and achieve the effects of good saccharification capacity and improved saccharification capacity.

Active Publication Date: 2020-06-30
SOUTHEAST UNIV
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Problems solved by technology

[0007] The technical problem to be solved in the present invention is to provide a kind of Trichoderma reesei genetically engineered bacteria with high cellulase production under the induction of carbon source, so as to solve the problems of low production of β-glucosidase and fiber production induced by soluble inducers in the prior art. Sulfase production low and other problems

Method used

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  • A strain of Trichoderma reesei genetic engineering bacteria with high cellulase production induced by soluble and insoluble carbon sources and its construction method and application
  • A strain of Trichoderma reesei genetic engineering bacteria with high cellulase production induced by soluble and insoluble carbon sources and its construction method and application
  • A strain of Trichoderma reesei genetic engineering bacteria with high cellulase production induced by soluble and insoluble carbon sources and its construction method and application

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Effect test

Embodiment 1b

[0050] Construction of embodiment 1bgl1-his recombinant plasmid

[0051] (1) bgl1-his gene amplification primers refer to the bgl1-linker-his primers in the summary of the invention (6). Using the cDNA of Trichoderma reesei Rut-C30 as a template, the bgl1-his gene was amplified with the primers. The amplification conditions are pre-denaturation: 95°C, 15s; denaturation: 95°C, 15s; annealing: 66°C, 30s; extension: 72°C, 3min; thorough extension: 72°C, 5min;

[0052] (2) The purified PCR product of the target gene was inserted into the XbaI restriction site of the plasmid pDht / sk by single-digestion homologous recombination to obtain the bgl1-his recombinant plasmid, see figure 1 ;

[0053] (3) adopting the method mediated by Agrobacterium to transfer the expression cassette into Trichoderma reesei spores, the specific method is as follows:

[0054] Agrobacterium tumefaciens transformation:

[0055] a. Agrobacterium tumefaciens maintained at -70°C were incubated on ice for 1...

Embodiment 2

[0065] The supernatants obtained from each strain of Trichoderma reesei cultured in cellulose medium shake flasks for 7 days were centrifuged at 4° C. at 8000 rpm for 15 minutes to collect the supernatants for the determination of cellulase activity. Cellulase activity assay method is as follows:

[0066] (1) Determination of β-glucosidase (pNPGase) enzyme activity: add 20 μL of appropriately diluted enzyme supernatant to 90 μl of 4 mM (50 mM NaCl, pH 5.0), incubate at 50 ° C for 10 min, draw 100 μ L and add an equal volume of 2% NaCO 3 , placed on a microtiter plate to read OD 405 (Biochem-Tokyo, 1999, 125(4):728-736). All enzyme activities are expressed as one enzyme activity unit IU per milliliter of fermentation broth. One enzyme activity unit (IU) is defined as: when p-nitrophenyl-β-D-galactopyranoside (pNPG) is used as substrate to release 1 μmol product p-nitrophenol (PNP) in 1 minute Enzyme amount.

[0067](2) Determination of exoglucanase (pNPCase) enzyme activity...

Embodiment 3

[0071] The culture supernatant obtained under different conditions was used to hydrolyze cellulose, and the cellulose hydrolysis efficiency and substrate specificity of the cellulase produced by Trichoderma reesei SEU-7 were detected. The specific method was as follows: Weigh the ethylenediamine-treated Corn stover (EDA-PCS) (Biotechnology for biofuels, 2015, 8:1-15) 75mg / mL was placed in a centrifuge tube, and 100mM sodium acetate buffer (pH 5.0) and 0.2mg / ml azide were added to each tube NaCl and 150 μg / mL crude enzyme solution. React at 50°C and 200rpm for 72h. The reducing sugar content was measured every 24 hours. The result is as Figure 5 It was shown that 11 mg / mL of glucose was produced after 72 h of cellulose hydrolysis, which was 2.6 times that of Rut-C30.

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Abstract

The invention relates to the field of gene engineering and microorganism fermentation, and discloses a trichoderma reesei genetically engineered bacterium SEU-7 for producing cellulase in high yield under induction of soluble and non-soluble carbon sources as well as a construction method and application; a classification name is Trichoderma reesei and a strain number is SEU-7; the bacterium is preserved in China Center for Type Culture Collection, a preservation number is CCTCCM 2016492 and a preservation date is September 18, 2016. The invention further discloses a construction method of the genetically engineered bacterium and application of the genetically engineered bacterium to production of the cellulase under the induction of the different carbon sources. Compared with the prior art, the trichoderma reesei genetically engineered bacterium for producing the cellulase in the high yield is constructed by utilizing a homologous recombination technology; the trichoderma reesei genetically engineered bacterium has an extremely high cellulase production capability under the induction of the soluble and non-soluble carbon sources. Meanwhile, produced cellulase liquid has a saccharifying function better than that of Rut-C30 cellulase liquid.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a Trichoderma reesei genetic engineering bacterium capable of high-yielding cellulase under the induction of soluble and insoluble carbon sources, its construction method and application. Background technique [0002] Cellulose is a macromolecular polysaccharide polymer widely found in nature. Cellulase can be used to degrade cellulose into sugars fermentable by bacteria for the production of biofuels, bio-based fine chemical products and pharmaceuticals. Cellulase is a complex enzyme mixture mainly including endoglucanase, exoglucanase and β-glucosidase. The complete degradation of cellulose requires the synergistic action of these three enzymes. Endoglucanase acts on the non-crystalline region of cellulose to degrade cellulose into cellooligosaccharides of different lengths, and then cellobiohydrolase, exoglucanase, degrades cellooligosaccharides into fibers Disaccha...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N1/15C12N15/80C12N9/42C12N9/24C12R1/885
CPCC07K2319/21C12N9/2437C12N9/2445C12N9/2477C12N15/80C12Y302/01004C12Y302/01021C12Y302/01091
Inventor 林凤鸣李程程周乐
Owner SOUTHEAST UNIV
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